DESCRIPTION: The mechanism of insulin action is a central question in biology with important ramifications into many areas of human disease, including diabetes, obesity, atherosclerosis and hypertension. Studies of the nematode Caenorhabditis Elegans have provided important insight into insulin action. In this simple metazoan, mutations of the insulin/IGF-1 receptor homologue Daf-2 gene prolong life span, decrease metabolic activity and increase fat accumulation. This characteristic stage, referred to as the dauer stage, can be rescued by mutations of the Daf-16 gene. This suggests that Daf-16 is a negative regulator of insulin/IGF-1 receptor signaling in C. Elegans. Daf-16 encodes a forkhead transcription factor. The investigators have shown that FKHR, the closest mammalian homologue of Daf-16, is an insulin-regulated transcription factor. In hepatocytes, insulin phosphorylates FKHR on at least three different amino acid residues through a hierarchical cascade initiated by the serine-threonine kinase, Akt. Insulin-dependent phosphorylation inhibits the ability of FKHR to stimulate transcription of prototypic insulin-responsive genes like PEPCK and IGFBP-1, in addition to genes that regulate apoptosis. Based on the tissue distribution of the three mammalian Fkhr genes in mice (Fkhr, FkhrII, Afx), as well as their seemingly different phosphorylation patterns, the investigators propose to test the hypothesis that FKHR mediates insulin action in liver and beta cells. There are two specific aims in this proposal. In aim 1, the investigators will address the role of FKHR in insulin-dependent glucose production in vitro. The investigators will first ask whether hepatic glucose production can be regulated by introducing dominant negative or constitutively active mutant FKHRs into hepatocytes from either normal mice or insulin receptor-deficient mice. Next the investigators will address whether differences in the phosphorylation patterns of renal and hepatic FKHR proteins can account for the tissue-specific ability of insulin to suppress hepatic, but not renal gluconeogenesis. In aim 2, the investigators will introduce null alleles of FKHR in mice using conditional mutagenesis and ask whether selective ablation of FKHR in liver and/or beta cells can rescue diabetes in insulin resistant mice by reducing glucose output or preventing beta cell apoptosis. Conversely, the investigators will attempt to induce diabetes in mice by means of a constitutively active mutant FKHR, generated through a novel "knock-in" approach. These investigations will shed new light onto the mechanisms by which insulin resistance causes hyperglycemia, and disclose new approaches to the treatment of diabetes.